The c-kit receptor protein in the testis of green frog Rana esculenta: seasonal changes in relationship to testosterone titres and spermatogonial proliferation

Abstract

The green frog Rana esculenta is a seasonal breeder. The cyclic changes between almost arrested and highly activated spermatogenesis offer an ideal model to study basic mechanisms of spermatogenesis. In this study, we demonstrated, to our knowledge for the first time, c-kit receptor positive cells in the testis of this amphibian. The presence of c-kit receptor protein was confirmed by western blotting (Wb) analyses carried out in the testis during all the three main phases of the sexual cycle. The antibody recognized a band of about 150 kDa that was correlated with the positive staining in the germinal epithelium. The immunolabelling for c-kit receptor, evaluated by immunohistochemistry (IHC), was localized in I and II spermatogonia (SPG), in I and II spermatocytes, in both elongating spermatids and spermatozoa and in the Leydig cells. Furthermore, c-kit expression showed a seasonal pattern connected with both testicular and plasma profiles of testosterone during the reproductive cycle. The highest expression of c-kit receptor occurred during the reproductive period, when the testis exhibited the maximum concentration of testosterone. In this period, the mitotic activity of germ cell, assessed by both Wb and IHC analyses for proliferating cell nuclear antigen (PCNA), was intensive. Indeed, during the post-reproductive period, testosterone titres were the lowest and the expression of both PCNA and c-kit receptor protein in the testis, although present, is minor when compared with the reproductive phase. This evidence suggests that cell division can continue sufficiently to accumulate SPG for the next spring, when new germinal cells undergo multiplication. Finally, during the pre-reproductive period, testosterone levels begin to increase and mitotic activity of germinal epithelium is comparably enhanced. These events seem to precede the period of maximum stimulated spermatogonial proliferation, i.e. the reproductive period. These results suggest that the c-kit receptor may play a role in germ cell proliferation and provide a basis for future detailed investigation of regulatory factors of the proliferation of SPG.

Immunoreactivity of c-kit Receptor Protein during the Prehibernation Period in the Oviduct of the Chinese Brown Frog, Rana chensinensis

“…To test our immunohistochemical results, we also performed Western blot analysis of the c-kit protein in an oviducal extract of R. chensinensis, and the results indicated that the polyclonal antibody recognized a single protein of about 150 kDa in the oviduct tissue of this species. Similar evidence has also been reported in Rana esculenta; that is, the c-kit receptor was present in testicular tissues, and the c-kit antibody recognized a protein band of a similar size, approximately 150 kDa, in the germinal epithelium [29]. The most commonly used method to evaluate cellular proliferation is PCNA immunostaining.…”

“…Evidence has accumulated that the c-kit receptor plays a critical role in the development of the mammalian ovary, oogenesis, folliculogenesis, blastocyst implantation and uterine growth and repair [8,13,22,33]. Recently, the c-kit receptor has received increasing attention for its role in the reproductive function of lower vertebrates [18,29,41].…”

Study of the potential spermatogonial stem cell compartment in dogfish testis, Scyliorhinus canicula L.

“…Recently, Raucci and Di Fiore (2007) have shown that c-kit receptor protein is expressed in both types of spermatogonia distinguishable in Rana esculenta but with intensities varying according to the reproductive period. Although this study has shown, for the first time, that spermatogonia of elasmobranchs can express the c-kit receptor, its use as a marker of differentiating spermatogonia in the dogfish requires additional study.…”

Localization of hematopoietic cells in the bullfrog (Lithobates catesbeianus)

“…Several studies have identified anuran molecules by using mammalian/human antibodies (Cianciarullo et al 1999;Ong et al 1993;Raucci et al 2006;Almeida et al 2007;Azevedo et al 2007;Raucci and Di Fiore 2007), indicating that some conservation of antigenic protein has occurred through phylogenetic development. Some studies also corroborate the protein conservation between anurans and mammals.…”

“…In dorsal embryonic tissues such as the nervous system, notochord, and somites, Kao and Bernstein (1995) have identified a Xenopus gene (Xkl-1) whose predicted protein shows homology with mammalian c-kit. Moreover, c-kit + spermatogonia and spermatocytes have been described in Rana esculenta by using anti-human c-kit antibody (Raucci and Di Fiore 2007).…”